PD-1 and PD-L1 are diagnostic biomarkers and PD-L1 a promising therapeutic target in HER2-positive and triple negative normal-like feline mammary carcinoma subtypes

Abstract

Tumor microenvironment has gained great relevance due to its ability to regulate distinct 15 checkpoints mediators, orchestrating tumor progression. In order to understand the role of the PD-16 1/PD-L1 axis in cats with mammary carcinoma, serum PD-1 and PD-L1 levels were compared with 17 healthy controls and with serum CTLA-4 and TNF-α levels. PD-1 and PD-L1 expression was 18 evaluated in TILs and cancer cells, as the presence of somatic mutations. Results showed that serum 19 PD-1 and PD-L1 levels were significantly higher in cats with HER2-positive (p=0.017; p=0.032) and 20 triple negative (TN) normal-like mammary carcinomas (p=0.004; p=0.015). Besides, cats presenting 21 these tumor subtypes showed a strong positive correlation between serum PD-1, PD-L1, CTLA-4 and 22 TNF-α levels. In tumors, PD-L1 expression in cancer cells was significantly higher in HER2-positive 23 samples than in TN normal-like tumors (p=0.010), as the percentage of PD-L1-positive TILs (p=0.038). 24Results from the PD-L1 gene sequencing identified two heterozygous mutations in exon 4 (A245T, 25 3.8%; V252M, 42.3%) and one in exon 5 (T267S, 3.8%). In summary, results support that serum PD-1 26 and PD-L1 levels can be used as diagnostic biomarkers of HER2-positive and TN normal-like feline 27 mammary carcinomas and suggest that the development of monoclonal antibodies may be a good 28 therapeutic strategy. 29 30 31 1. Introduction 32 Feline mammary carcinoma (FMC) is the third most common tumor in the cat. As in human 33 breast cancer [1], FMC presents an aggressive and infiltrative behavior [2,3], with both HER2-positive 34 and triple negative (TN) subtypes showing worse prognosis than luminal A and B subtypes. Thus, 35 the identification of novel diagnostic biomarkers and therapeutic targets is needed, not only to 36 improve the clinical outcome of cats with mammary carcinoma but also because FMC shares 37 clinicopathological features and molecular classification with human breast cancer [1,4]. 38In humans, the role of the PD-1/PD-L1 axis is being investigated in different tumor types, with 39 the PD-L1 expressed by tumor cells regulating T cell activity, promoting immune suppression and 40 tumor escape. Recently, a monoclonal antibody that blocks PD-L1 binding to the PD-1 receptor on T 41 cells was approved by the Food and Drug Administration (FDA) to treat different tumor types and 42 also PD-L1 positive unresectable locally advanced and metastatic TN breast cancer [5,6]. The less 43 studied serum PD-1 (sPD-1) and PD-L1 (sPD-L1) levels may also play an important role in anti-tumor 44 immune responses. Studies in patients with breast cancer, lung cancer, metastatic melanoma and 48 inhibits T-cell immune function, being also targeted in breast cancer. Some trials are evaluating the 49 combination of anti-PD-1/PD-L1 antibodies with CTLA-4 inhibitors [5,10]. In humans, CTLA-4 50 regulates T-cell proliferation at the initial stage of immune responses, primarily in lymph nodes, 51whereas PD-1 suppresses T cells in later stages, typically in p...